1. Field of the Invention
The present invention is direct to the field of immunology. Specifically, the present invention provides methods for proliferating and differentiating B cells.
2. Related Art
During an immune response, activation and differentiation of B cells lead to the secretion of high affinity antigen-specific antibodies. When the antibody response is directed against protein antigens the production of antibodies is also dependent on the activation of specific helper T (Th) cells and their interactions with B cells. During the processes of activation and differentiation, B cells follow one or more distinct and irreversible pathways to acquire one of several identifiable functionalities. For example, initially activated B cells undergo high levels of proliferation, and during this period of cell division several different events can occur: isotope switching can occur to give recombination of heavy chain variable (V) region genes proximal to different constant regions (.gamma. subclasses, .alpha., or .epsilon.) and secretion of different antibody classes; somatic mutation can occur in the heavy and light chain V region gene segments to generate high affinity antibody combining sites; and B cells can differentiate to become memory cells or efficient antibody secreting plasma cells.
Which pathway(s) a particular B cell follows is most likely determined by the types of stimuli it receives; this is presumably determined by its overall milieu. For example, the presence of other cell types (different types of Th cells and/or follicular dendritic cells) and the presence or absence of specific antigen may deliver different signals to B cells. Some events, such as the signals that generate memory B cells and the molecular mechanisms that drive somatic mutation and selection of high affinity antibody producing cells, are poorly understood. Other processes, such as initial activation signals that drive naive B cells to proliferate and which are provided by contact with specific primed and activated Th cells have now been well characterized. (Parker, D., Annu. Rev. Immunol. 11:331-360 (1993)). Additionally, the effects of soluble Th cell-derived lymphokines on activated B cells have also been delineated (Parker, D., Annu. Rev. Immunol. 11:331-360 (1993); Hodgkin et al., J. Immunol. 145:2025-2034 (1990); Noelle et al., J. Immunol. 146:1118-1124 (1991)).
Because the generation of primed and activated Th cells is obligatory for B cell activation, defining the requirements of B cells themselves using classical in vitro systems employed for studying B cell responses has been difficult. In these systems, many of which utilized purified B cells, antigen-specific Th cell clones, and antigen, requirements for Th cell activation also were essential for generating a B cell response.
I. Contact-Dependent Delivery of Signals fiom Th Cells to B Cells
The steps involved in B cell activation by Th cells can be divided into first, the major histocompatibility complex (MHC)-restricted activation and priming of specific Th cells, and second, the interaction of secondary activated Th cells with B cells to induce B cell activation, proliferation, and differentiation. (Parker, D., Annu. Rev. Immunol. 11:331-360 (1993)). Naive Th cells are primed by interactions wizth antigens that have been cleaved into peptides (processed) which have bound to class II MHC molecules on the surface of antigen presenting cells. At the Th cell priming stage, resting B cells are not able to serve as antigen presenting cells because they do not appear to be capable of providing necessary costimulatory signals to the Th cells. Antigen presenting cells that can provide appropriate costimulatory signals for Th cell priming are interdigitating dendritic cells found in the T cell rich areas of the spleen and possibly activated B cells. Once specific Th cells are primed, their activation requirements appear to be less stringent. Primed Th cells can be efficiently activated by resting B lymphocytes that have captured antigen with specific antigen receptors and processed it to peptides. (Parker, D., Annu. Rev. Immunol. 11:331-360 (1993)). Once activated, primed Th cells express genes that enable them to reciprocally deliver appropriate contact- and Iymphokine-dependent activation signals to resting B cells. (Kehry, M. R., and Hodgkin, P. D., Sem. Immunol. 5:393-400 (1993)).
II. Historical Perspective
Several studies in the 1980's described critical features of specific signals delivered by activated primed Th cells to B cells. (Coffman et al., Immunol. Rev. 102:5-28 (1988); DeFranco et al., J. Exp. Med. 159:861-880 (1984)). B cell-Th cell contact was required; the Th cell-derived signals that drive resting B cell activation could not be replaced by soluble lymphokines alone (Andersson et al., Proc. Natl. Acad. Sci. USA 77:1612-1616 (1980); Julius et al., Proc. Natl. Acad. Sci. USA 79:1989-1993 (1982); Owens, T., Eur. J. Immunol. 18:395-401 (1988); Whalen et al., J. Immunol. 141:2230-2239 (1988); Hirohata et al., J. Immunol. 140:3726-3744 (1988); Noelle et al., J. Immunol. 140:1807-1814 (1989); Julius et al., Eur. J. Immunol. 18:381-386 (1988)). A finding important in characterizing the molecules involved in delivering contact signals to resting B cells was that preactivated Th cells could be employed to deliver activation signals. This implied that the contact signals received by naive B cells did not need to be cognate; they were antigen independent and were not MHC restricted. The B cell activating signals appeared to be different from signals required for Th cell activation and were thus capable of generating significant bystander B cell responses. (Whalen et al., J. Immunol. 141:2230-2239 (1988)). This suggested that the B cell receptors for Th cell-dependent contact signals were constitutively expressed and were not polymorphic or related to the B cell antigen receptor. Additionally, the Th cell molecules delivering the contact signals were most likely not functionally present in resting Th cells. This rules out a role for the T cell receptor complex that was initially involved in receiving antigen-specific Th cell activation signals.
These studies also delineated a role for Th cell-derived lymphokines in determining the amount and isotope of antibody secreted. (Coffman et al., Immunol. Rev. 102:5-28 (1988)). In the mouse, the ability of a Th cell clone to induce B cell differentiation has been shown to depend on the repertoire of lymphokines secreted after its activation. Th2 type Th cell clones, that secrete IL-4 and IL-5, are effective in inducing production of IgM and switching to IgG1, IgA, and IgE (Coffman et al., Immunol. Rev. 102:5-28 (1988); Mosmann & Coffman, Annu. Rev. Immunol. 7:145-173 (1989)). Although some controversy initially existed about the ability of Th1 type Th cell clones, that produce IL-2 and IFN-.gamma., to stimulate B cell differentiation, it appears that most Th1 cell clones are capable of providing the appropriate contact and soluble signals to activate resting B cells to secrete antibody when Th2 lymphokines are provided. (Abbas et al., J. Immunol. 144:2031-2037 (1990)).
Although the role of lymphokines seemed to be inducing B cell differentiation and not in initiating activation events, a system that separately delivered Th cell contact from soluble signals was needed. An early study demonstrated that membrane components from T cells could be transferred to other cell types by a Sendai virus-mediated fusion event. (Lindqvist et al., Scand. J. Immunol. 23:119-125 (1986)). These cells contained functional T cell-specific membrane proteins that could transduce signals for the activation of T cell-specific genes, but the cells were not examined for the ability to activate B cells. An additional study demonstrated that tumor cell plasma membranes were sufficient to generate allogeneic cytotoxic T lymphocytes (Stallcup et al., Cell. Immunol. 89:144-150 (1984)). This suggested that cell-cell contact provided critical signals that regulated lymphocyte activation. However, high doses of plasma membranes were nonspecifically inhibitory to both T and B lymphocyte responses (Stallcup et al., Cell. Immunol. 89:144-150 (1984)). Several years later, Sekita et al. prepared plasma membranes from a variety of T cell and lymphoid cell lines and demonstrated their ability to stimulate and costimulate B cell proliferation (Sekita et al., Eur. J. Immunol. 18:1405-1410 (1988)). However, these investigators found that membranes prepared from most cell types could activate B cells (Sekita et al., Eur. J. Immunol. 18:1405-1410 (1988)). Since no specificity was observed for Th cells or for activated over resting T cells, the active component in those membranes appeared to be different from that induced by activation of normal primed Th cells.
Studies that showed the most promise for developing a Th cell-free system for delivering contact signals to B cells used a plasma membrane-enriched fraction of vesicles prepared from an activated mouse Th2 cell clone, D10.G4.1 (D10), to deliver contact signals in the absence of other cell types (Brian, A. A., Proc. Natl. Acad. Sci. USA 85:564-568 (1988)). In this work, specificity was demonstrated in that membranes prepared from resting Th2 cells were not stimulatory for B cell proliferation. However, the B cell population had not been purified to eliminate preactivated B cells, which have different activation requirements than resting B cells. Preactivated B cells respond to Th2 lymphokines alone (in particular, IL-5) by growing and secreting IgM in the absence of Th cell-dependent contact signals (Takatsu et al., Immunol. Rev. 102:107-135 (1988)). Because it is not possible to separate contaminating endoplasmic reticulum vesicles that would contain lymphokines from plasma membrane vesicles it was not clear in the initial work (Brian, A. A., Proc. Natl. Acad. Sci. USA 85:564-568 (1988)) whether an actual contact-dependent stimulus could be delivered to resting B cells by a plasma membrane fraction from a Th cell clone.